Here we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic " Lab-on-a-Disc " cartridge. Isolation of white blood... more
Here we present retrieval of Peripheral Blood Mononuclear Cells by density-gradient medium based centrifugation for subsequent analysis of the leukocytes on an integrated microfluidic " Lab-on-a-Disc " cartridge. Isolation of white blood cells constitutes a critical sample preparation step for many bioassays. Centrifugo-pneumatic siphon valves are particularly suited for blood processing as they function without need of surface treatment and are 'low-pass', i.e., holding at high centrifugation speeds and opening upon reduction of the spin rate. Both 'hydrostatically' and 'hydrodynamically' triggered centrifugo-pneumatic siphon valving schemes are presented. Firstly, the geometry of the pneumatic chamber of hydrostatically primed centrifugo-pneumatic siphon valves is optimised to enable smooth and uniform layering of blood on top of the density-gradient medium; this feature proves to be key for efficient Peripheral Blood Mononuclear Cell extraction. A theoretical analysis of hydrostatically primed valves is also presented which determines the optimum priming pressure for the individual valves. Next, 'dual siphon' configurations for both hydrostatically and hydrodynamically primed centrifugo-pneumatic siphon valves are introduced; here plasma and Peripheral Blood Mononuclear Cells are extracted through a distinct siphon valve. This work represents a first step towards enabling on disc multi-parameter analysis. Finally, the efficiency of Peripheral Blood Mononuclear Cells extraction in these structures is character-ised using a simplified design. A microfluidic mechanism, which we termed phase switching, is identified which affects the efficiency of Peripheral Blood Mononuclear Cell extraction.
There is increasing evidence that, in addition to their presence, the propensity of circulating tumour cells to form multi-cellular clusters bears significant information about both cellular resistance to chemotherapy and overall... more
There is increasing evidence that, in addition to their presence, the propensity of circulating tumour cells to form multi-cellular clusters bears significant information about both cellular resistance to chemotherapy and overall prognosis. We present a novel two-stage, stopped-flow, continuous centrifugal sedimentation strategy to measure the size distributions of events (defined here as cells or clusters thereof) in a blood sample. After off-chip removal of red blood cells, healthy white blood cells are sequestered by negative-immunocapture. The purified events are then resolved along a radially inclined rail featuring a series of gaps with increasing width, each connected to a designated outer collection bin. The isolation of candidate events independent of target-specific epitopes is successfully demonstrated for HL60 (EpCAM positive) and sk-mel28 (EpCAM negative) cells using identical protocols and reagents. The propensity to form clusters was quantified for a number of cell lines, showing a negligible, moderate or elevated tendency towards cluster formation. We show that the occupancy distribution of the collection bins closely correlates with the range of cluster sizes intrinsic to the specific cell line.
We present a substantially improved design and functionality of a centrifugo-magnetophoretic platform which integrates direct immunoseparation and cost-efficient, bright-field detection of cancer cells in whole blood. All liquid handling... more
We present a substantially improved design and functionality of a centrifugo-magnetophoretic platform which integrates direct immunoseparation and cost-efficient, bright-field detection of cancer cells in whole blood. All liquid handling takes place in a disposable cartridge with geometry akin to a conventional compact disc (CD). The instrumentation required to process such a "lab-on-a-disc" cartridge can be as simple and cost-efficient as the rotor on a common optical disc drive. In a first step, target cells in a blood sample are specifically bound to paramagnetic microbeads. The sample is then placed into the disc cartridge and spun. In the second step, magnetically tagged target cells are separated by a co-rotating, essentially lateral magnetic field from the background population of abundant blood cells, and also from unbound magnetic beads. A stream of target cells centrifugally sediments through a stagnant liquid phase into a designated detection chamber. The contin...
Research Interests:
The faithful replication of DNA and the accurate segregation of genomic material from one generation to the next is critical in the maintenance of genomic stability. This chapter will describe the structure and assembly of an... more
The faithful replication of DNA and the accurate segregation of genomic material from one generation to the next is critical in the maintenance of genomic stability. This chapter will describe the structure and assembly of an epigenetically inherited locus, the centromere, and its role in the processes by which sister chromatids are evenly segregated to daughter cells. During the G2 phase of the cell cycle kinetochores are assembled upon the chromatids. During mitosis, kinetochores attach chromosome(s) to the mitotic spindle. The kinetochore structure serves as the interface between the mitotic spindle and the chromatids and it is at the kinetochore where the forces that drive chromatid separation are generated. Unattached chromosomes fail to satisfy the spindle assembly checkpoint (SAC), resulting in cell cycle arrest. The centromere is the locus upon which the kinetochore assembles, and centromeres themselves are determined by their unique protein composition. Apart from budding yeast, centromeres are not specified simply by DNA sequence, but rather through chromatin composition and architecture and are thus epigenetically determined. Centromeres are built on a specific nucleosome not found elsewhere in the genome, in which histone H3 is replaced with a homologue - CENP-A or CenH3. This domain is flanked by heterochromatin and is folded to provide a 3-dimensional cylinder-like structure at metaphase that establishes the kinetochore on the surface of the mitotic chromosomes. A large family of CENtromere Proteins (CENPs) associates with centromeric chromatin throughout the cell cycle and are required for kinetochore function. Unlike the bulk of histones, CENP-A is not assembled concurrently with DNA synthesis in S-phase but rather assembles into the centromere in the subsequent G1 phase. The assembly of CENP-A chromatin following DNA replication and the re-establishment of this network of constitutive proteins have emerged as critical mechanisms for understanding how the centromere is replicated during the cell cycle.
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests:
Research Interests: Kinetics, Cell Cycle, DNA replication, DNA damage, DNA repair, and 15 moreDNA, Cell line, Humans, Protein Kinases, Tetracycline, Phosphorylation, Histones, Cell nucleus, DNA damage response, UV irradiation, Protein Kinase, Human Fibroblasts, Cell Cycle Proteins, DNA Polymerase, and Biochemistry and cell biology
Research Interests:
Research Interests:
The human skin cancer-prone disease xeroderma pigmentosum variant (XPV) results from a mutation in RAD30, which encodes the novel lesion bypass DNA polymerase eta. XPV cells are characterized by delayed completion of DNA replication and... more
The human skin cancer-prone disease xeroderma pigmentosum variant (XPV) results from a mutation in RAD30, which encodes the novel lesion bypass DNA polymerase eta. XPV cells are characterized by delayed completion of DNA replication and increased mutagenesis following UV irradiation. In cell-free extracts of XPV lymphoblasts, functional DNA polymerase eta is required for the complete replication of a double-stranded plasmid containing either a single (6-4) photoproduct or a cyclobutane pyrimidine dime(CPD), the major mutagenic UV-induced lesion. In cultured XPV cells, replication arrest activates downstream signalling pathways, leading to hyperphosphorylation of the 34-kDa subunit of the trimeric single-stranded DNA-binding protein, RPA (replication protein A). Many of the RAD30 mutations identified in XPV cells result in truncation and inactivation of DNA polymerase eta. To examine whether polymorphisms in the RAD30 gene that result in altered polymerase eta function, rather than enzyme inactivation, might contribute to individual susceptibility to skin cancer, methods to screen for sequence changes in the RAD30 gene in human genomic DNAhave been developed.